Chapters 22, 24, 25, 9, 11 and 12

Biochemistry Exam 3

What is semiconservative DNA replication?

Each daughter DNA molecule receives one parental strand and one newly synthesized strand

What is an origin of replication?

A specific AT-rich DNA sequence where replication begins.

What is a replication fork?

The region where parental DNA unwinds and new DNA is synthesized on both strands.

Why is the leading strand continuous while the lagging strand is discontinuous?

DNA polymerase only synthesizes 5'→3', requiring Okazaki fragments on the lagging strand.

What does helicase do?

Unwinds the DNA double helix.

What does primase do?

Synthesizes short RNA primers needed for DNA polymerase to begin synthesis.

What do SSB proteins do?

Prevent re-annealing of separated DNA strands.

What does DNA polymerase I do?

Removes RNA primers and replaces them with DNA.

What does DNA polymerase III do?

The major enzyme responsible for replicating the bacterial chromosome.

What does DNA ligase do?

Seals nicks between Okazaki fragments by forming phosphodiester bonds.

What does topoisomerase do?

Relieves supercoiling tension during replication.

What problem occurs when replicating the 5’ ends of linear chromosomes?

The RNA primer at the extreme 5’ end cannot be replaced, creating a shortening problem.

What is the function of telomerase?

Extends telomeres using its own RNA template to prevent chromosome shortening.

What polymerase is used in PCR and why?

Thermostable Taq polymerase from thermophiles, able to withstand high temperatures.

What are the 3 steps of PCR?

Denaturation (98°C), annealing (~55°C), extension (72°C).

What is a dideoxynucleotide (ddNTP)?

A nucleotide lacking the 3’-OH group, used in Sanger sequencing to terminate DNA synthesis.

How do you read a Sanger sequencing gel?

From bottom (shortest fragments = 5’ end) to top, identifying which ddNTP terminated each fragment.

What is the role of RNA polymerase σ-factor?

Recognizes promoter sequences and initiates transcription.

What is the transcription bubble?

Unwound DNA region where RNA polymerase synthesizes RNA.

What happens to mRNA in bacteria after synthesis?

It has a very short half-life (~2–3 minutes) and degrades quickly.

What are the three molecular players in translation?

mRNA, ribosomes, and tRNA

In which direction is mRNA read?

5’→3’

In which direction is the polypeptide synthesized?

N-terminus → C-terminus

What are the three stop codons?

UAA, UGA, UAG

What is the start codon and what does it code for?

AUG → methionine (fMET in prokaryotes)

What is degeneracy of the genetic code?

Multiple codons can encode the same amino acid

What is the function of aminoacyl-tRNA synthetaase?

Attaches the correct amino acid to corresponding tRNA

What is the Shine-Dalgarno sequence?

Ribosome-binding site in prokaryotic mRNA ~ 10 bases upstream of AUG

What are the ribosomal subunits in bacteria vs. eukaryotes?

Bacteria: 30S + 50S = 70S

Eukaryotes: 40S + 60S = 80S

What is the general formula for carbohydrates?

(CH2O)n

What is a monosaccharide?

A simple sugar (3-9 carbons)

What is an oligosaccharide?

A few linked monosaccharides (e.g. disaccharidess)

What is a polysaccharide?

A long chain polymer of monosaccharides

What is a glycan?

General term for oligo- and polysaccharides

How do you determine D- vs. L- configuration?

Look at the chiral center farthest from the carbonyl:

-OH right = D

-OH left = L

What is an epimer?

Two sugars differing at exactly one chiral carbon

What is an anomer?

Isomers differing at the anomeric carbon (α vs β after ring closure)

What is mutarotation?

Interconversion between α and β anomers in solution

What is a hemiacetal?

Formed when a carbonyl reacts with an alcohol; basis of sugar cyclization.

What is the difference between furanose and pyranose?

Furanose = 5-membered ring;
Pyranose = 6-membered ring.

Which ring conformation is most stable for hexoses?

The chair conformation.

What is a reducing sugar?

A sugar with a free anomeric carbon capable of reducing Cu²⁺.

What test identifies reducing sugars?

Fehling’s/Benedict’s/Tollens – blue → brick-red.

What happens when a sugar is reduced?

Carbonyl → sugar alcohol (alditol), e.g., sorbitol.

What happens when a sugar is oxidized at C1?

Forms an aldonic acid.

What happens when a sugar is oxidized at C6?

Forms a uronic acid

What are amino sugars?

Sugars containing amino groups; e.g., GlcNAc.

How is a glycosidic bond formed?

Condensation reaction (loss of water) between anomeric carbon and an –OH group.

What determines disaccharide structure?

• Monomers involved

• Carbon positions linked

• Order of sugars

• α or β configuration

What is amylose?

Unbranched α(1→4) glucose polymer of starch.

What is amylopectin?

Branched α(1→4) glucose with α(1→6) branches.

What is glycogen?

Highly branched glucose storage polymer in animals.

What is cellulose?

β(1→4) glucose polymer; structural component of plants.

What is chitin?

Polymer of N-acetylglucosamine; found in arthropod exoskeletons.

What are glycosaminoglycans (GAGs)?

Long repeating disaccharides with amino sugars; e.g., heparin, hyaluronic acid.

What is peptidoglycan composed of?

NAG and NAM with peptide crosslinks.

How does penicillin work?

Inhibits transpeptidase → stops crosslinking in peptidoglycan.

Difference between Gram+ and Gram– bacteria?

Gram+: thick peptidoglycan, retains stain;
Gram–: thin peptidoglycan + outer membrane.

What is an N-linked glycoprotein?

Carbohydrate attached to Asn.

What is an O-linked glycoprotein?

Carbohydrate attached to Ser/Thr.

What determines ABO blood type?

Specific O-linked oligosaccharides on RBC surfaces.

What does influenza hemagglutinin (HA) do?

Binds host receptors to allow viral entry.

What does neuraminidase (NA) do?

Cleaves sialic acid to release new viral particles.

How does Tamifluwork?

Inhibits neuraminidase, blocking viral release.

Intermediary metabolism 

Synthesis (anabolism) and degradation (catabolism) of small molecules (metabolic intermediates)

Energy metabolism

Pathways that generate or store energy (ATP, NADH, FADH₂).

What are central pathways?

Pathways with the highest metabolic traffic and energy transfer

Why are central pathways important?

Highly conserved across organisms

ex. glycolysis, citric acid cycle, oxidative phosphorylation, fatty acid oxidation/synthesis, gluconeogenesis, photosynthesis

Why is ATP hydrolysis highly exergonic?

• Resonance stabilization of products (ADP + Pi).

• Electrostatic repulsion between phosphate groups in ATP.

• Greater solvation of products vs. reactants.

• ΔG°’ ≈ -32.2 kJ/mol.

How does ATP coupling make unfavorable reactions favorable?

• Couples endergonic reactions (positive ΔG°’) to ATP hydrolysis (negative ΔG°’).

  • Example: F-6-P + Pi → F-1,6-BP (ΔG°’ = +16.3 kJ/mol) becomes favorable when coupled to ATP → ADP.

What makes carbonyl carbons good electrophiles?

• Polarized C=O bond → carbon is electron-poor.

• Adjacent atoms (C or H) are poor leaving groups, favoring addition reactions.

• Common in aldehydes, ketones, esters, carboxylic acids.

List common biochemical nucleophiles.

Hydroxide (OH⁻), alkoxide (RO⁻), carbanion (C⁻), thiolate (RS⁻), amines (R-NH₂), imidazole (His side chain), carboxylate (R-COO⁻)

SN1

• Leaving group departs → carbocation intermediate.

• Nucleophile attacks carbocation.

• Results in racemic mixture (loss of stereochemistry).

SN2

• Nucleophile attacks simultaneously as leaving group departs.

• Pentavalent transition state.

• Results in inversion of stereochemistry.

What is nucleophilic acyl substitution?

• Nucleophile attacks acyl carbon → tetrahedral oxyanion intermediate.

• Intermediate collapses → leaving group expelled.

• Examples: ester hydrolysis, peptide bond cleavage (chymotrypsin).

What is carbonyl condensation?

• Enolate ion (resonance-stabilized) attacks carbonyl carbon.

• Forms new C–C bond.

• Aldol condensation: enolate + aldehyde/ketone.

• Claisen condensation: enolate + ester.

What is β-elimination in metabolism?

• Removal of H and OH from β-hydroxycarbonyl → double bond formation.

• Example: dehydration of 2-phosphoglycerate → phosphoenolpyruvate (PEP).

How do oxidation-reduction reactions work with NAD⁺?

• NAD⁺ accepts hydride ion (H⁻, 2e⁻ + 1H⁺).

• NADH donates hydride in reductive reactions.

• Enzymes: dehydrogenases, oxidases, reductases.

What are the two phases of glycolysis and their purposes?

• Energy investment phase (steps 1–5): Glucose phosphorylated and split into two triose phosphates. Consumes 2 ATP.

• Energy generation phase (steps 6–10): Oxidation of GAP → ATP + NADH. Produces 4 ATP, 2 NADH.

Step-by-step: Reaction 1 (Hexokinase): mechanism and regulation?

• Glucose + ATP → Glucose-6-phosphate (G6P).

• Nucleophilic substitution: glucose OH attacks γ-phosphate of ATP.

• ΔG°’ = -18.4 kJ/mol.

• Regulation: inhibited by product (G6P). Isoforms differ in Km (Hexokinase I–III low Km, Hexokinase IV/glucokinase high Km).

Step-by-step: Reaction 2 (Phosphoglucose isomerase): why necessary?

• G6P → F6P.

• Converts aldose to ketose.

• Prepares for symmetric cleavage in aldolase step.

• ΔG°’ = +1.7 kJ/mol (near equilibrium).

Step-by-step: Reaction 3 (PFK): regulation details?

• F6P + ATP → FBP.

• ΔG°’ = -15.9 kJ/mol.

• Major control point of glycolysis.

• Allosteric regulation:

  • Activated by AMP, F-2,6-BP.

  • Inhibited by ATP, citrate.

Step-by-step: Reaction 4 (Aldolase): mechanism?

• FBP → DHAP + GAP.

• ΔG°’ = +23.9 kJ/mol (driven forward in vivo).

• Mechanism: Schiff base (imine) formation with Lys residue → retro-aldol cleavage.

Step-by-step: Reaction 5 (Triose phosphate isomerase): importance?

• DHAP ↔ GAP.

• ΔG°’ = +7.6 kJ/mol.

• Ensures both triose phosphates enter glycolysis.

• Enzyme is diffusion-limited (“perfect enzyme”).

Step-by-step: Reaction 6 (GAPDH): coupling logic?

• GAP + NAD⁺ + Pi → 1,3-BPG + NADH.

• ΔG°’ = +6.3 kJ/mol.

• Coupled oxidation (exergonic) with acyl phosphate formation (endergonic).

• Conserves energy in high-energy intermediate.

Step-by-step: Reaction 7 (Phosphoglycerate kinase): energy conservation?

• 1,3-BPG + ADP → 3PG + ATP.

• ΔG°’ = -17.2 kJ/mol.

• Substrate-level phosphorylation.

• Net ΔG for reactions 6+7 = -10.9 kJ/mol.

Step-by-step: Reaction 8 (Phosphoglycerate mutase): role?

• 3PG → 2PG.

• ΔG°’ = +4.4 kJ/mol.

• Prepares substrate for elimination.

Step-by-step: Reaction 9 (Enolase): type of reaction?

• 2PG → PEP + H₂O.

• ΔG°’ = -3.2 kJ/mol.

• α,β-elimination → high-energy PEP.

Step-by-step: Reaction 10 (Pyruvate kinase): why strongly exergonic?

• PEP + ADP → Pyruvate + ATP.

• ΔG°’ = -29.7 kJ/mol.

• Exergonic due to enol → keto tautomerization.

• Regulation: activated by F-1,6-BP, inhibited by ATP.

Aerobic fate of pyruvate

Pyruvate → Acetyl-CoA → TCA cycle → oxidative phosphorylation. NADH oxidized in mitochondria

Anaerobic fate of pyruvate

• Homolactic fermentation: Pyruvate → Lactate (regenerates NAD⁺).

• Alcoholic fermentation: Pyruvate → Acetaldehyde → Ethanol + CO₂ (regenerates NAD⁺)

Which glycolysis steps are irreversible and regulated?

Hexokinase, PFK, Pyruvate kinase.

• Large negative ΔG.

• Allosteric regulation prevents futile cycles.

• Different signals regulate glycolysis vs. gluconeogenesis

How does reciprocal regulation prevent futile cycles?

• Glycolysis enzyme (PFK) activated by AMP/F-2,6-BP.

• Gluconeogenesis enzyme (FBPase)